Chromatin fibers

Nucleus The nucleus is separated from the cytosol by a double membrane, the nuclear envelope. The DNA is complexed with basic proteins (histones) to form chromatin fibers, the material from which chromosomes are made. A distinct RNA-rich region, the nucleolus, is the site of ribosome assembly. The nucleus is the repository of genetic information encoded in DNA and organized into chromosomes. During mitosis, the chromosomes are replicated and transmitted to the daughter cells. The genetic information of DNA is transcribed into RNA in the nucleus and passes into the cytosol where it is translated into protein by ribosomes. 

Tohno et al. (20) used similar steps to determine the chromatin loop size in human leukemia cells (an ultrahigh-resolution EM was not needed). To determine chromatin loop size, the lengths of 102 chromatin fibers protruding from four nuclei were measured and their average lengths were estimated. The steps they used (essentially those of Miller and Bakken (21) are summarized as follows ... 

During the cell cycle, chromosome structures shuttle between de-condensed interphase and condensed mitosis states. Dynamic changes also occur at the lower levels of architectures, i.e., at the chromatin and nucleosome levels. Upon gene activation and inactivation, folding and unfolding of the nucleosome structure and the chromatin fibers occur at limited loci of the genome. Namely, the structures of the chromosome are dynamic and mobile. Nevertheless, there are basic structural units that remain stable and constitute the fundamental chromosome architecture. 

The effect of the superhelical strain of the DNA template on the nucleosome structure can be investigated from the in vitro chromatin reconstitution system (for the detail of in vitro chromatin reconstitution, see sections 2.1 and 2.3). Interestingly, the efficiency of the reconstitution becomes higher as the lengths of the DNA used are longer (Hizume et al, 2004) (Fig. 3a-c). In the 3 kb reconstituted chromatin, one nucleosome could be formed in every 826 bp DNA on average, while in the 106 kb chromatin fibers, one nucleosome can be formed in every 260 bp of DNA. The chromatin reconstituted on the any length of linearized plasmid, the efficiency of the reconstitution becomes one nucleosome per 800 bp DNA. The treatment of the... 

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

In order to investigate the structural and functional characteristics of the chromatin fiber, several methods for in vitro nucleosome reconstitution have been developed (Lusser and Kadonga, 2004). Among them, the salt-dialysis method is the simplest... 

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

It has been assumed that the nucleus contains an immobile structure , in which chromatin fibers are partially attached and fixed. These structures are called nuclear matrix or nuclear scaffold . When cells are successively treated with detergent, high-salt solution and DNase I, the nuclear scaffold can be observed as a fibrous network in the cell nucleus (Fey et al, 1986 Nickerson, 2001 Yoshimura et al, 2003) (Fig. 2b). The biochemical analyses of the nuclear scaffold have identified a 174kDa protein as a major component (Fisher et al, 1982). This protein is now known as topo II (Berrios et al, 1985). 

Figure 5. (Continued) different panels (e and f for HeLa, j and k for chicken erythrocyte, and o and p for yeast). A section profile obtained along X-Y line shows a typical granular structure in die nucleus (e, j, o), and the peak-to-peak distance between the granular structure was distributed from 60 nm to 120 nm (e). The diickness of the chromatin fibers released out of die nucleus varied possibly due to the assembly of diinner fibers (f, k, p). A section profile for the spread fibers was obtained along X-Y line (f, k, p). Isolated HeLa cell nucleus was treated widi (r, s) or without (q) RNase. The treatment releases SOnmfiber from the nucleus. The histogram of die fiber width is shown in an inset of (s). Bars, 250 nm. (See Colour Plate 2.)...

Adolph KW, Kreisman LR, Kuehn RL (1986) Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy. Biophys J 49 221-231... 

Hizume K, Yoshimura SH, Maruyama H, Kim J, Wada H, Takeyasu K (2002) Chromatin reconstitution development of a salt-dialysis method monitored by nano-technology. Arch Histol Cytol 65 405 13 Hizume K, Yoshimura SH, Takeyasu K (2004) Atomic force microscopy demonstrates a critical role of DNA superhelicity in nucleosome dynamics. Cell Biochem Biophys 40 249—262 Hizume K, Yoshimura SH, Takeyasu K (2005) Linker histone HI per se can induce three-dimensional folding of chromatin fiber. Biochemistry 44 12978-12989 Hofmann WA, de Lanerolle P (2006) Nuclear actin to polymerize or not to polymerize. J Cell Biol 172 495-496... 

Karymov MA, Tomschik M, Leuba SH, Caiafa P, Zlatanova J (2001) DNA methylation-dependent chromatin fiber compaction in vivo and in vitro requirement for hnker histone. Faseb J 15 2631—2641 Kaszas E, Cande WZ (2000) Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin. J Cell Sci 113(Pt 18) 3217-3226... 

Simpson RT, Thoma F, Brubaker JM (1985) Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones a model system for study of higher order structure. Cell 42 799-808 Sugiyama S, Yoshino T, Kanahara H, Kobori T, Ohtani T (2003) Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes. Scanning 25 132-136 Sugiyama S, Yoshino T, Kanahara H, Shichiri M, Fukushi D, Ohtani T (2004) Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy. Anal Biochem 324 39 4... 

Luger K, Hansen JC (2005) Nucleosome and chromatin fiber dynamics. Curr Opin Struct Biol 15 188-196... 

Kepert J.F Mazurkiewicz J, Heuvelman GL, Toth KF, Rippe K (2005) NAPl modulates binding of linker histone HI to chromatin and induces an extended chromatin fiber conformation. J. Biol. Chem 280 34063-34072... 

Hansen JC (2002) Conformational dynamics of the chromatin fiber in solution determinants, mechanisms, and functions. Annu Rev Biophys Biomol Struct 31 361-392... 

Hansen JC (2002) Conformational dynamics of the chromatin fiber in solution determinants, mechanisms, and functions. Annu Rev Biophys Biomol Struct 31 361-392 Hassan AH, Prochasson P, Neely KE, Galasinski SC, Chandy M, Carrozza MJ, Workman JL (2002) Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell 111 369-379... 

The chapter is organized as follows. Section II is devoted to materials and methods. In Section III, we show [34, 35] that the GC content displays rather regular nonlinear oscillations with two main periods of 110 20 kbp and 400 50 kbp, which are well-recognized characteristic scales of chromatin loops and loop domains involved in the hierarchical folding of chromatin fibers. 

---